This project is designed to continue studies of marcomolecular binding, biosynthesis and action of steroids. Affinity-labeling steroids will be used to define the comparative topography (structure) of the steroid binding sites of several high affinity steroid binding proteins. These include four enzymes (crystalline human placental estradiol 17 beta-dehydrogenase, homogeneous 20 beta-hydroxysteroid dehydrogenase from S. hydrogenans, and estradiol 17 beta- and 17 alpha-dehydrogenases from sheep placenta), three receptors (estrogen receptor from calf uterus; estrogen and progesterone receptor from chick oviduct) and one transport protein (Testosterone-Estradiol Binding Globulin from human plasma). Certain questions of endocrinologic and biochemical importance will also be addressed, using these models. Further, subunit analysis, sequencing, X-ray crystallography and other molecular methods of analysis will be carried out in an extensive study of the structure and catalytic mechanism of the human placental enzyme. This will, we think, validate affinity labeling as a method of structural analysis. The ultimate goals are: a) define the intimate structural relationships of steroid, cofactor and amino acid residues of human placental estradiol 17 beta-dehydrogenase pertinent to the catalytic event; b) validate affinity labeling; c) find out what steroid binding proteins have in common in terms of structure of the steroid binding site; d) discover how evolution has provided for the catalytic removal of epimeric 17-hydrogens; 3) elucidate the extent of active site modification which permits continued catalytic activity and the role of the enzyme in orientation of steroid substrate and cofactor; f) determine whether steroids covalently bound to receptor have biological activity in vitro (which rules out intranuclear steroid transfer to another macromolecule and biologically validates affinity labeling).